FvMYB79 Positively Regulates Strawberry Fruit Softening via Transcriptional Activation of FvPME38

Strawberry is a soft fruit with short postharvest life, due to a rapid loss of firmness. Pectin methylesterase (PME)-mediated cell wall remodeling is important to determine fruit firmness and softening. Previously, we have verified the essential role of FvPME38 in regulation of PME-mediated strawberry fruit softening. However, the regulatory network involved in PME-mediated fruit softening is still largely unknown. Here, we identified an R2R3-type MYB transcription factor FvMYB79, which activates the expression level of FvPME38, thereby accelerating fruit softening. During fruit development, FvMYB79 co-expressed with FvPME38, and this co-expression pattern was opposite to the change of fruit firmness in the fruit of ‘Ruegen’ which significantly decreased during fruit developmental stages and suddenly became very low after the color turning stage. Via transient transformation, FvMYB79 could significantly increase the transcriptional level of FvPME38, leading to a decrease of firmness and acceleration of fruit ripening. In addition, silencing of FvMYB79 showed an insensitivity to ABA-induced fruit ripening, suggesting a possible involvement of FvMYB79 in the ABA-dependent fruit softening process. Our findings suggest FvMYB79 acts as a novel regulator during strawberry ripening via transcriptional activation of FvPME38, which provides a novel mechanism for improvement of strawberry fruit firmness.


Introduction
Fruit softening is a major determinant of shelf life and commercial value. It is the consequence of multiple cellular processes, including changes in soluble sugar content, glycosylation of cellulose, and remodeling of cell wall structure. Fruit cell walls are highly enriched in pectin, occupying more than 50% of the wall materials [1]. Pectin is the most abundant class of macromolecule within the cell wall matrix [2]. Pectin is also abundant in the middle lamellae between primary cell walls, where it functions as major adhesions between cells. During fruit ripening, a range of pectin-degrading enzymes are secreted into the cell wall to modify pectin structure, resulting in pectin polymer degradation [3]. Pectin methylesterase (PME) catalyzes reactions on pectin de-methylesterification to generate carboxyl groups during the release of methanol and hydrogen ions. PME provides the hydrolysis substrate for polygalacturonase (PG), and functions synergistically with the PG enzyme to soften fruit [4]. Previous studies have found that PME activity significantly increased associated with the ripening and softening process of various fruits, including banana, mango, strawberry, and kiwi fruit [5][6][7][8]. During tomato fruit ripening, lower PME activity was associated with an increased content of methylesterification of pectin, a decreased level of total and chelator soluble polyuronides in cell walls, as well as a more soluble solids contain. Despite the decrease of PME enzyme activity in ripening

FvMYB79 Are Transcriptionally Correlated with FvPME38 during Strawberry Fruit Development
In our previous study, FvPME38 and FvPME39 regulated fruit softening at the maturity stage of strawberry fruit [12], while the upstream regulatory network of FvPME38 and FvPME39 was still unknown. To determine the complex regulatory network, we used the fruitENCODE database [33] to clarify DNA methylation, accessible chromatin and histone modifications of FvPME38 and FvPME39 genes during strawberry fruit development. Based on the speculation from 5 methylation cyanine (5mC) and DNaseI hypersensitive sites (DHS), from immature stage to ripe stage, the signal of 5mC significantly decreased, but DHS signal remarkably increased in the promoter region of the two PME genes, suggesting that the promoters of FvPME38 and FvPME39 are demethylated and became accessible only in the stage of ripening fruit ( Figure 1A). In contrast, immature strawberry fruit contained higher hyper-H3K27me3 in the FvPME38 loci ( Figure 1A). To identify the possible upstream regulator of FvPME38 and FvPME39, 1.5 kb promoter region was captured and analyzed by PlantCARE. Bioinformatics analysis indicated that the cis-elements in FvPME38 and FvPME39 promoters mainly contained the phytohormone-responsive motifs and TF binding sites ( Figure 1B). These findings implied that the transcriptional level of FvPME38 and FvPME39 genes are controlled by diverse regulators including phytohormones and TFs.

FvMYB79 Are Transcriptionally Correlated with FvPME38 during Strawberry Fruit Development
In our previous study, FvPME38 and FvPME39 regulated fruit softening at the maturity stage of strawberry fruit [12], while the upstream regulatory network of FvPME38 and FvPME39 was still unknown. To determine the complex regulatory network, we used the fruitENCODE database [33] to clarify DNA methylation, accessible chromatin and histone modifications of FvPME38 and FvPME39 genes during strawberry fruit development. Based on the speculation from 5 methylation cyanine (5mC) and DNaseI hypersensitive sites (DHS), from immature stage to ripe stage, the signal of 5mC significantly decreased, but DHS signal remarkably increased in the promoter region of the two PME genes, suggesting that the promoters of FvPME38 and FvPME39 are demethylated and became accessible only in the stage of ripening fruit ( Figure 1A). In contrast, immature strawberry fruit contained higher hyper-H3K27me3 in the FvPME38 loci ( Figure 1A). To identify the possible upstream regulator of FvPME38 and FvPME39, 1.5 kb promoter region was captured and analyzed by PlantCARE. Bioinformatics analysis indicated that the cis-elements in FvPME38 and FvPME39 promoters mainly contained the phytohormoneresponsive motifs and TF binding sites ( Figure 1B). These findings implied that the transcriptional level of FvPME38 and FvPME39 genes are controlled by diverse regulators including phytohormones and TFs.  In order to search for the upstream regulators of FvPME38 and FvPME39, we obtained 609 TF members from bHLH, NAC, WRKY, MYB, bZIP and MADS gene family which have binding cis-elements in FvPME38 and FvPME39 promoters (Table S1). Co-expression network analysis based on the transcriptome data from fruitENCODE database showed that 27 TFs were identified as positive or negative co-expressed members with FvPME38 or FvPME39 (Figure 2A and Table S2). Their expression level were further examined in the five different stages of 'Ruegen' fruit by q-PCR assay. Among them, FvWRKY55, FvMYB65, FvMYB79 and FvNAC114 were expressed highest at ripening stage, which coincided with the expression pattern of FvPME38 and FvPME39 ( Figure 2B). We further individually measured the FvMYB79 transcript in achenes and receptacles of 'Ruegen' (red fruit) and 'Hawaii-4' (white fruit). The expression level of FvMYB79 was both significantly up-regulated in achenes and receptacles at turning at the ripening stage which reached the highest point at the last stage of Ruegen fruit, but, in contrast, strongly decreases in both receptacles and achenes of Hawaii-4 from stage RS2 to RS4 ( Figure 2C-F). Accompanying fruit development, the change tendency of FvMYB79 transcript and fruit firmness was negatively correlated, implying that FvMYB79 may be involved in regulation of strawberry fruit firmness.
In order to search for the upstream regulators of FvPME38 and FvPME39, we obtained 609 TF members from bHLH, NAC, WRKY, MYB, bZIP and MADS gene family which have binding cis-elements in FvPME38 and FvPME39 promoters (Table S1). Coexpression network analysis based on the transcriptome data from fruitENCODE database showed that 27 TFs were identified as positive or negative co-expressed members with FvPME38 or FvPME39 (Figure 2A and Table S2). Their expression level were further examined in the five different stages of 'Ruegen' fruit by q-PCR assay. Among them, FvWRKY55, FvMYB65, FvMYB79 and FvNAC114 were expressed highest at ripening stage, which coincided with the expression pattern of FvPME38 and FvPME39 ( Figure 2B). We further individually measured the FvMYB79 transcript in achenes and receptacles of 'Ruegen' (red fruit) and 'Hawaii-4' (white fruit). The expression level of FvMYB79 was both significantly up-regulated in achenes and receptacles at turning at the ripening stage which reached the highest point at the last stage of Ruegen fruit, but, in contrast, strongly decreases in both receptacles and achenes of Hawaii-4 from stage RS2 to RS4 ( Figure 2C-F). Accompanying fruit development, the change tendency of FvMYB79 transcript and fruit firmness was negatively correlated, implying that FvMYB79 may be involved in regulation of strawberry fruit firmness.  S3, S5, S7, RS2 and RS4 were described previously [12,18]. (D,F) Spatial and temporal expression level of FvMYB79 gene during 'Ruegen' and 'Hawaii 4' fruit development in achenes and receptacles. Error bars represent SD of three independent replicates.

FvMYB79 Is an ER and Nucleus-Localized Transcriptional Factor
Sequence analysis of FvMYB79 showed that it has a complete open reading frame (ORF) region (507 bp in length), which encodes a protein with 168 amino acids (GenBank accession number MN530978) with a calculated molecular mass of 19.38 kDa and isoelectric point of 10.44. To predict the function of FvMYB79, a phylogenetic tree was constructed using the amino acid sequences of FvMYB79 and MYBs from other plant species, which are well known to control phenylpropanoid, anthocyanins and proanthocyanins biosynthesis ( Figure 3A). Although FvMYB79 was clustered with the known anthocyanin-and proanthocyanin-associated MYB TFs, including production of anthocyanin pigment 1(PAP1), production of anthocyanin pigment 2 (PAP2), TRANSPARENT TESTA 2(TT2) from Arabidopsis [34][35][36], MdMYB10 from Malus x domestica [37], anthocyanin 2(AN2) from Petunia hybrida [38], VvMYBPA2 from Vitis vinifera [39], LjTT2a and LjTT2b from Lotus japonicus [40], MtMYB14 from Medicago truncatula [41], the bootstrap value of FvMYB79 was lower than 50 precent, indicating a potential different function of FvMYB79 ( Figure 3A).  To examine the subcellular localization of the FvMYB79 protein, FvMYB79 was fused with N-terminal of green fluorescent protein (GFP) tag, driven by the CaMV 35S promoter. Visualization of FvMYB79-GFP by transient transformation in tobacco leaf cells showed that the FvMYB79-GFP signal was co-localized with the well-characterized endoplasmic reticulum (ER) marker tdTomato-HDEL, as well as the nucleus marker mRFP-NLS. Hence, FvMYB79 has a dual localization in both ER and nucleus ( Figure 3B).
The amino acid sequence alignment analysis showed that FvMYB79 protein contains To examine the subcellular localization of the FvMYB79 protein, FvMYB79 was fused with N-terminal of green fluorescent protein (GFP) tag, driven by the CaMV 35S promoter.
Visualization of FvMYB79-GFP by transient transformation in tobacco leaf cells showed that the FvMYB79-GFP signal was co-localized with the well-characterized endoplasmic reticulum (ER) marker tdTomato-HDEL, as well as the nucleus marker mRFP-NLS. Hence, FvMYB79 has a dual localization in both ER and nucleus ( Figure 3B).
The amino acid sequence alignment analysis showed that FvMYB79 protein contains typical R2-and R3-MYB DNA-binding domains, and a highly conserved Lx2[R/K]x3Lx6Lx3R motif for interaction with other bHLH proteins [42][43][44] (Figure 3C). Previous studies showed that the EAR motif was able to convert a transcriptional activator into a strong repressor [45]. Furthermore, there isn't ERF-associated amphiphilic repression domain (EAR) in the highly divergent C-terminal region of FvMYB79 ( Figure 3C), suggesting that FvMYB79 may act as a transcriptional activator.

FvMYB79 Regulates Strawberry Fruit Softening via Transcriptional Activation of FvPME38
To further elucidate the functionality of FvMYB79, transient overexpression and RNA interference (RNAi) of FvMYB79 were performed in 'Ruegen' fruit at 18 d after pollination (DAP). After infiltration, a dramatic delay of fruit ripening was found in FvMYB79 RNAi fruits, but FvMYB79 overexpressed fruits accelerated the ripening process ( Figure 4A-C). Measurement of fruit rigidity showed that FvMYB79 RNAi fruits has higher firmness but overexpression of FvMYB79 reduced fruit firmness, indicating that FvMYB79 influences fruit rigidity ( Figure 4C,D).
To test the regulatory network of FvMYB79, we firstly examined the interaction of FvMYB79 with potential partners in the MBW complex and other genes encoding regulatory proteins associated with fruit development and ripening. A Y2H assay showed that FvMYB79 was not able to interact with the proteins that are crucial for fruit ripening, including MADS9, SHP, SnRK2.6, RIF, TTG1, bHLH33 and bHLH3 ( Figure S3). To search for the other downstream regulators of FvMYB79, some flavonoid and softening related genes were selected, of which expression levels were regulated by FvMYB79 ( Figure 4E,F). We cloned the 2.0 kb promoter sequences of them to perform dual-luciferase assay. The results indicated that FvMYB79 activated with FvPME38 promoter in a specific manner ( Figure 4G). To further investigate whether FvMYB79 could bind directly to the promoter of FvPME38 yeast, one-hybrid assay was carried out. The self-activation activity of the promoter was screened with aureobasidin A (AbA), and the results showed that 30 ng·mL −1 AbA could inhibit the growth of yeast cells. Yeast one-hybrid screens indicates that yeast cells co-transformed with FvMYB79-effector and pFvPME38-report grew on SD/-Leu medium (with 0, 20× and 30× AbA), demonstrating that FvMYB79 could bind directly to the promoter of FvPME38 ( Figure 4H). Altogether, FvMYB79 plays a role in the regulation of strawberry fruit softening by controlling the transcriptional level of FvPME38.

FvMYB79 Regulates Strawberry Fruit Softening via Transcriptional Activation of FvPME38
To further elucidate the functionality of FvMYB79, transient overexpression and RNA interference (RNAi) of FvMYB79 were performed in 'Ruegen' fruit at 18 d after pollination (DAP). After infiltration, a dramatic delay of fruit ripening was found in FvMYB79 RNAi fruits, but FvMYB79 overexpressed fruits accelerated the ripening process ( Figure 4A-C). Measurement of fruit rigidity showed that FvMYB79 RNAi fruits has higher firmness but overexpression of FvMYB79 reduced fruit firmness, indicating that FvMYB79 influences fruit rigidity ( Figure 4C,D).

FvMYB79 Might Be Involved in Abscisic Acid-Dependent Fruit Ripening Process
Endogenous ABA has been proved essentially for the onset of ripening process in strawberry fruit [50]. In our previous study, the expression level of FvPME38 is positively regulated by ABA [12]. We thus speculated that FvMYB79-FvPME38 module might participate in ABA-dependent fruit ripening. We applied endogenous ABA or nordihydroguaiaretic acid (NDGA, an ABA inhibitor) on the 'Ruegen' fruits at the turning red stage, and then quantitatively examined fruit ripening by calculation of the percentages of different stages of fruits and measurement of fruit firmness. In comparison with the control group, exogenous ABA treatment significantly accelerated fruit ripening, displaying higher anthocyanin accumulation and reduced fruit firmness, whereas NDGA-treated fruits postponed anthocyanin accumulation and fruit softening ( Figure 5A-C). Correspondingly, NDGA treatment significantly reduced and ABA treatment stimulated the transcript abundance of FvMYB79 transcript ( Figure 5D). In RNAi-FvMYB79 fruits, the ABA effect on the promotion of fruit ripening was significantly delayed (Figure 5E-I). We further detected ABA content in FvMYB79 RNAi fruits. The endogenous ABA content was dramatically reduced in FvMYB79 RNAi fruits, in comparison to control ( Figure 5J). We therefore postulated that FvMYB79 may be involved in the regulation of ABA-dependent fruit ripening. These results also suggested that FvMYB79 accelerates the fruit ripening process possibly mediated by the ABA signal.

Identification of Regulator Combining Epigenetics, Transcriptomics and Co-Expression Analysis
Strawberry ripening is a highly coordinated program which leads to structural and bio-chemical changes, such as sudden receptacle softening and increase in the contents of sugars, anthocyanins, volatile compounds, and vitamins. The loss of firmness of receptacles is one of the main processes in the onset of fruit ripening due to the activity of cell wall remodeling enzymes. In our previous study, FvPME38 and FvPME39 regulated fruit softening in the onset of strawberry fruit ripening, and showed expression levels which were positively regulated by ABA [12]. Although these studies help to understand the biological function of PME when fruit softening, the complex regulatory network involved in the process remains to be further explored. Global transcriptome and co-expression network analyses are becoming increasingly used to infer gene function, and set common pathways and putative targets for TFs in many perennial plants which are difficult to use knockout or knockdown T-DNA mutant methods to verify gene function because of the long generation time [51,52]. In the present study, we provided a high-efficiency way to identify and predict the function of candidate genes genome-wide combining epigenetics, transcriptomics and co-expression analysis. Using known FvPME38 and FvPME39 genes as bait (target) genes, DNA methylation, accessible chromatin and histone modifications of those genes were further analysed to find out the sequence region of dramatic change of epigenetics signals during fruit development, and predicting cis-elements on the sequence region of those genes. Afterwards, all members of the probable TF gene family were identified or obtained from published database before. The combination of expression profiles from transcriptome data and physiological trait analysis paves the way to quickly select candidate genes correlated with the target trait. The findings from fruitENCODE [33] and PlantCARE [53] database showed that the transcriptional level of FvPME38 and FvPME39 genes are controlled by various factors, including phytohormones and TFs, during plant developmental processes. Further co-expression network analysis with transcriptome data showed that 27 TF genes from bHLH, NAC, WRKY, MYB, bZIP and MADS families were identified as positive or negative co-expression members with FvPME38 or FvPME39. The expression level of candidate genes were further examined in the five different stages of 'Ruegen' fruit by q-PCR assay. FvWRKY55, FvMYB65, FvMYB79 and FvNAC114 were expressed highest at ripening stage, which coincided with the expression pattern of FvPME38 and FvPME39. Among them, FvNAC114, namely FaRIF in previous study, was reported as a key regulator of strawberry fruit ripening [49]. In 'Ruegen' (red fruit) and 'Hawaii-4' (white fruit), the expression level of FvMYB79 was both significantly up-regulated in achenes and receptacles after the ripening stage, which reached its highest at the last stage of Ruegen fruit, but, in contrast, strongly decreases in both receptacles and achenes of Hawaii-4 from stage RS2 to RS4. It suggests that, in the two genotypes, the function of FvMYB79 was similar at the early stage of fruit ripening, but was different at the late stage. Finally, we functionally characterized a R2R3-type MYB, FvMYB79, which has not been described so far. Molecular and phenotypic analyses of transient FvMYB79-overpression and FvMYB79-RNAi fruits indicated that FvMYB79 promotes strawberry fruit softening through activating the transcriptional level of FvPME38 ( Figure 5K), but did not result in altered expression levels of hormonal, sugar and acid pathway related genes.

FvMYB79 Responses ABA Signal to Specially Promote Strawberry Fruit Softening
Different phytohormones have been established involving strawberry fruit growth and ripening processed [14]. Auxin is mainly produced in achenes, while abscisic acid (ABA), bioactive free base cytokinins, gibberellins, and ethylene are synthesized predominantly in receptacles [14]. Endogenous ABA content is low at early stages and gradually increases to control a series of fruit ripening programs, such as fruit softening and the accumulation of flavonoids, sucrose and acid [19]. Based on our results, we propose that ABA and FvMYB79 may act in a positive regulatory feedback loop to promote strawberry fruit ripening. Firstly, transcript levels of FvMYB79 are substantially diminished after application of NDGA, while significantly increased after ABA treatment. Exogenous application of ABA in fruits significantly promotes the ripening process, which could be delayed in FvMYB79-RNAi fruits. Those results indicated that ABA acts upstream of FvMYB79. Secondly, we found that ABA content significantly decreased in FvMYB79 RNAi fruits when compared with control, revealing that FvMYB79 acts upstream of ABA biosynthesis. This regulatory mechanism is similar with other TFs, such as FaRIF, which is involved in a feedback regulation loop with ABA to regulate the strawberry ripening process [49].
Combining analyses of molecular and phenotypic experiment, our results showed that FvMYB79 activates the transcriptional level of FvPME38 gene to specially promote strawberry fruit softening, but is not involved in the accumulation of flavonoid, sucrose and acid during strawberry fruit development. Transient overexpression or RNAi at the expression level of FvMYB79 at the turning stage of 'Rugen' strawberry significantly changed transcriptional levels of softening and flavonoid-related genes, but the expression level of hormonal, sugar and acid related genes were not significantly changed in the transient transformed fruits. Previous studies showed that FvMYB10 and FvMYB1 interact with FvbHLH3/33 and/or FvTTG1 (WD40 protein) to form a ternary MYB-BHLH-WD40 (MBW) complex, which is a positive and negative regulator of anthocyanins and flavonoid accumulation, respectively [26][27][28]. However, unlike FvMYB10 and FvMYB1, FvMYB79 didn't interact with FvTTG1 and/or FvbHLH3/33 ( Figure S3). Furthermore, the overexpression of FvMYB79 in 'Hawaii-4' fruits (white fruit) didn't promote anthocyanin accumulation in the transient transformed fruits. Dual-luciferase assay and yeast one-hybrid assay analysis showed that FvMYB79 only significantly activates the promoter of FvPME38, but didn't activate the promoter of flavonoid related genes, such as MYB10, DFR and UFGT. Altogether, these results demonstrated that FvMYB79 plays an important role for the regulation of strawberry fruit ripening and softening by controlling the transcriptional level of FvPME38. In a previous study, we observed a dramatic delay of fruit ripening in FvPME38 RNAi fruits, but overexpression of the FvPME38 accelerated ripening process [12], which in accord with the phenotype of transient overexpression or RNAi of FvMYB79 in 'Ruegen' fruits. The function of FvPME38 was similar with other cell wall-modifying genes, such as FvXTH9, FvXTH6 and FaβGAL4. Overexpression of these genes led to faster ripening by modification of the cell wall components in strawberry fruits [54,55]. Although FvMYB79 acts upstream of ABA biosynthesis, we speculate that the promoting fruit ripening in FvMYB79 overexpressed 'Ruegen' fruits was not caused by ABA signal. Because endogenous ABA content controls a series of fruit ripening programmed, such as fruit softening, accumulation of flavonoid, sucrose and acid [19], but FvMYB79 only regulates fruit softening process without a change in the transcriptional level of hormonal, sucrose and acid related genes ( Figure S1). A previous study showed that pectin demethylesterification by PME is the main source of plant-derived methanol [56], and methanol is crucial in control of plant growth and response to stresses [57][58][59]. Therefore, FvMYB79 controls transcriptional level of FvPME38, which may determine the production of methanol via cell wall pectin demethylesterification to regulate strawberry fruit ripening.

Plant Materials
The seventh generation inbred lines of F. vesca accessions, namely Ruegen (Ru F7-4, red-fruited) and Hawaii 4 (PI551572, white-fruited) were used as wild-types in this study. The plants were grown in a greenhouse (16 h/8 h light conditions at 22 • C, at a relative humidity of 65%). The samples, used for RNA isolation, were frozen in liquid nitrogen immediately after collection and then stored at −80 • C.

Identification of Cis-Element on Promoters of FvPME Genes
DNA methylation, dynamic chromatin accessibility, and histone modification of FvPME genes loci in immature fruit and fully ripened strawberry fruit tissues were obtained from the fruitENCODE website [33]. Cis-elements on the 1.5 kb promoter sequences of FvPME genes were predicted by PlantCARE [53]. The position of the cis-elements were displayed using an online website: Gene Structure Display Server (GSDS) [60].

Co-Expression Network
RNA-seq data of strawberry was gained from the fruitENCODE website [33]. Pearson's correlation coefficient (PCC) values were used as a measurement of expression similarity between gene pairs, and filtered with Excel software (parameter was set as >0.9). Visualizations of the data were carried out by Cytoscape software [61].

Gene Expression Analysis by qRT-PCR
cDNA, used for quantitative reverse transcription-PCR (q-PCR) analysis, was synthesized using one-step genomic DNA removal and a cDNA synthesis kit (Transgen, Beijing, China). Q-PCR was performed using the MonAmp TM ChemoHS qPCR Mix (Monad, Wuhan, China). Primers were shown in Table S3. The analysis was performed using three biological samples and three technical repeats. Relative expression levels of each gene were normalized to an internal control Fvactin and FvGAPDH by 2 −∆∆Cp algorithm [62].

Phylogenetics, Gene Structure and Motif Analyses
A phylogenetic tree was constructed using MEGA X [63] with neighbor-joining (NJ) criteria and verified using the maximum likelihood (ML) method, and 1000 bootstrap replicates were performed based on the multiple alignments of the full-length amino acid sequences using ClustalW [64].

Texture Analyses
TA.XT.plus Texture Analyser (Stable Micro Systems Ltd., Surrey, UK) along with the measuring probe P/5S (5 mm Spherical stainless steel, supplied with the Texture Analyser) were employed for texture determination. The system was equipped with texture profile analysis (TPA). Hardness was measured as the maximum penetration force (N) reached during tissue breakage. Measurable parameters were: pretest speed 1 mm·s −1 ; test speed 1 mm·s −1 penetrating distance of 5 mm into the fruit. The measurement was triggered automatically at 0.04 N.

Plasmid Construction
The primers used for plasmid construction are listed in Table S3. The coding region of FvMYB79 gene was amplified from the cDNA of 'Ruegen' strawberry using PrimerSTAR ® GXL DNA Polymerase (TaKaRa, Maebashi, Japan), sub-cloned into pDONR221, and then inserted into the binary vector pK7WG2D using Gateway ® Technology. For RNAi, the partial coding sequence of FvMYB79 (301-504 bp) was sub-cloned into pDONR221, and then inserted into the binary vector pK7WIWG2D. The correct fusion constructs were transferred into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method.

Subcellular Localization
The coding region of FvMYB79 gene was sub-cloned into pDONR221, and then inserted into the binary vector pGWB605 to form the fusion vector 35S:FvMYB79-GFP using Gateway ® Technology. The leaves of three-week-old Nicotiana benthamiana plants were infiltrated through their abaxial surfaces by Agrobacterium suspension (OD 600 = 0.6). At 72 h post-infiltration, a fluorescence signal was visualized using a Zeiss LSM880 confocal microscope (Zeiss, Oberkochen, Germany).

Transient Transformation of Strawberry Fruit Flesh
Transiently transformed strawberry fruit flesh was carried out using agro-infiltration as previously described [65]. GV3101 strains which harbor FvMYB79 overexpression or RNAi constructs was infiltrated into the 'Ruegen' or 'Hawaii-4' fruit flesh at 18 d after pollination (DAP) using syringes. Six plants or fruits were injected with each construct in triplicates. The transformed samples were placed in the dark at 22 • C overnight and then transferred to a phytotron (22 • C, 16-h of light and 8-h darkness) for seven days. The GFP signal of fruit was examined using the fluorescence dissecting stereomicroscope (Nikon, Tokyo, Japan). Pictures were taken and tissues were collected for downstream analysis.

Yeast Two-Hybrid Assay
A yeast two-hybrid (Y2H) assay was performed using the Matchmaker ® Gold Yeast Two-Hybrid System (Clontech, http://www.clontech.com/ (accessed on 10 September 2020). The coding region of MYB79, MADS9, SHP, SnRK2.6, RIF, TTG1, bHLH3 and bHLH33 were amplified using the primers listed in Table S3 and individually inserted into pGBKT7 bait vector or pGADT7 prey vector, respectively. The bait and prey constructs were co-transformed into yeast strain Y2H Glod according to the manufacturer's instructions (Yeastmaker, Clontech, Mountain View, CA, USA). Y2H Glod cells were grown on SD/-Trp-Leu medium for at least three days. Positive colonies were confirmed by PCR and further grown on SD/-Trp-Leu-His-Ade medium.

Dual-Luciferase Reporter Assays
The dual luciferase assay was carried out according to previous report [66]. The promoter sequences were amplified from 'Ruegen' DNA and inserted into the pGreenII 0800-LUC vector to construct the reporter constructs. All these promoter constructs were individually transformed into A. tumefaciens strain GV3101 that contains the pSoup helper plasmid using the freeze-thaw method. Agrobacterium containing the effector vector or report vector were re-suspended separately with infiltration buffer (10 mM MgCl 2 ; 200 µM acetosyringone; 10 mM MES, pH 5.5) and mixed in a ratio of 9:1 to a final concentration of OD 600 0.9-1. After that the mixed cultures incubated at 25 • C with 60 r.p.m. shaking for two hours before infiltration. Two-week-old N. benthamiana leaves were infiltrated with the mixed bacterial cultures using needleless syringes. Three days after infiltration, firefly luciferase (LUC) and renilla luciferase (REN) were assayed using dual-luciferase assay reagents (Promega, Madison, WI, USA).

Yeast One-Hybrid Assay Analysis
The CDS of FvMYB79 and promoters of FvPME38 were cloned and inserted into the pGADT7 vector and the pABAi vector, respectively. Then, the bait plasmid was linearized and integrated into Y1H Gold yeast (Clontech, http://www.clontech.com (accessed on 10 September 2020)), and tested the promoter autoactivation on SD/-Ura agar plate containing Aureobasidin A(AbA, 0, 100, 200, 300, 400, 500, 600, 800, 1000 ng/mL). Furthermore, the interaction between FvMYB79 and the promoters of FvPME38 were detected on SD/-Leu medium with the optimum AbA concentration. The empty pGADT7 of the corresponding recombinant pABAi was used as the negative control.

Hormonal Treatment Assay of Strawberry Fruits
The concentration of hormone solution is in accordance with a previous study [14]. The concentration of stock solution was 50 mM and 5 mM in ethanol for abscisic acid (ABA) and nordihydro-guaiaretic acid (NDGA) (Aladdin, Beijing, China), respectively. The concentration of working solution for treatment were 1 mM for ABA and 100 µM for NDGA diluted in ddH 2 O. GV3101 strains which harbor FvMYB79 RNAi constructs was infiltrated into the 'Ruegen' fruit flesh at 18 DAP using syringes. At the same time, about 20 µL ABA working solution was injected into the 18 DAP fruits.

Measuring Fruit ABA Content
Fruit Sample powder was dissolved in 900 µL ethyl acetate and 100 µL [ 2 H6-ABA] (CSA number: 35671-08-0, OlChemIm, Olomouc, Czech Republic) internal standard (concentration: 100 ng/mL). Next, the samples were vortexed for 30 s and sonicated in 4 • C for 20 min. After centrifugation at 12,000 rpm for 3 min at 4 • C, the supernatant was obtained and then evaporated until dry using a vacuum concentrator (Labconco, America). The dried residues were diluted in 200 µL of 70% methanol at 4 • C and then vortexed for 10 s and sonicated in 4 • C for 5 min. After centrifugation at 12,000 rpm for 2 min at 4 • C, the supernatant was obtained and then filtered through a 0.22 mm PVDF membrane. ABA was separated and detected using high-performance liquid chromatography (Acquity UPLC, Waters, Milford, MA, USA) coupled with a triple quadrupole mass spectrometer (XEVO TQS MS, Waters, Milford, MA, USA).

Statistical Analysis
Statistical analysis was done by one-way ANOVA in the Graphpad prism 8.0 software (Graphpad Software, San Diego, CA, USA). Significant differences between groups were calculated using p < 0.05 in one-way ANOVA analysis, Tukey's HSD post hoc test.

Conclusions
Our study identified an R2R3-type MYB transcription factor, FvMYB79, via epigenetics, transcriptomics and co-expression multi-analysis. We verified FvMYB79 functions as a sensor of the ABA signal involved in the strawberry fruit softening process via transcriptional activation of FvPME38. Our findings shed light on the transcriptional regulatory mechanisms underlying hormone-mediated fruit softening to provide preliminary knowledge for improving strawberry fruit quality.
Author Contributions: C.X. and X.C. conceived and designed the project. C.X., J.C. and X.M. performed experiment. C.W. prepared fruit samples. C.X. and J.C. performed the data analysis. C.X. wrote the manuscript. X.C. and Z.G. provided guidance for experimental design and data analysis, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.